Adeno-associated virus (AAV) is a promising delivery system for gene therapy. However, current manufacturing of AAV suffers from very low yields compared to other biotherapeutics. The AAV dose per patient ranges between 10 11and 10 15 viral genomes (vg), requiring an average of 10 to 30 L production/dose. As a consequence, production costs are prohibitive for most indications. Our recent studies revealed that only 10% of the HEK293 cells that have received the AAV encoding DNA produce assembled AAV capsids. This observation prompts the question: Why would cells that have been successfully transfected, be unable to produce AAV. To answer this question, we undertook a detailed study to characterize the two sub-populations from the same transfection, the cells that were making assembled capsids and those that were not. We found that the two populations had distinct cell cycle profiles, with a block in cell cycle progression characterizing the producer population. RNA-seq analysis of the two populations reveals differences in the molecular pathways impacted and provides a basis for making changes to improve productivity.